Method of producing protease by microorganism

ABSTRACT

Certain microorganisms of Bacillus, Candida and Rhodotorula produce protease when cultured on a medium containing an organic acid as the predominant source of carbon.

United States Patent Niwa et a1.

[ 1 June 13, 1972 METHOD OF PRODUCING PROTEASE BY MICROORGANISM [72]Inventors: Kunimori Niwa, 2-160, Kannon, Kawasakishi; Hiroshiro Shibai,10-2-204, Tsujidodanchi, Fujisawa-shi; Masahiro Yasunaga, 5, Zenbu-cho,Hodogaya-ku, Yo k0hamashi; Yoshio Hirose, 1155, Nakamaruko,Kawasaki-shi; Teruo Shiro, 2-2-28, Matsunami, Chigasaki-shi, all ofKanagawa-ken,.lapan [22] Filed: Sept. 23, 1970 [21] App1.No.: 74,831

[30] Foreign Application Priority Data Oct. 3, 1969 Japan ..44/79033[52] U.S. Cl. ..195/66 R [51] Int.Cl. ..Cl2d 13/10 [58] FieldofSearch.... 195/65, 30, 66, 62, 114, 117,195/1 18 [56] References CitedUNITED STATES PATENTS 3,411,990 11/1968 Udagawa et a1 ..l95/3O X3,616,234 10/1971 Komagata et a1. ..195/66 R Primary Examiner-Lionel M.Shapiro Attorneyl(elman and Berman 57 ABSTRACT Certain microorganisms ofBacillus, Candida and Rhodotorula produce protease when cultured on amedium containing an organic acid as the predominant source of carbon.

5 Claims, No Drawings METHOD OF PRODUCING PROTEASE BY MICROORGANISM Thepresent invention relates to a method of producing protease by means ofmicroorganisms.

It is well known that protease is produced by microorganisms cultured ona medium containing as the carbon source, such carbohydrates as starch,glucose or starch hydrolyzate.

It has now been found that a microorganism of genus Bacillus Candida andRhodotorula when cultured on a medium containing an organic acid as thesole or principal carbon source, can produce protease in very highyield.

Microoragnisms capable of growing on culture media which contain anorganic acid as the sole or principal carbon source and capable ofproducing protease include Bacillus subtilis AJ-3l68FERM P-242),Bacillus sp AJ-3208(NRRL B3700), Bacillus sp AJ-3205 (NRRL 3-3699),Candida lipolytica A.l-4555(ATCC 16617), Candida lipolytica ATCC 16618and Rhodotorula glutinis AJ-5193(NRRL Y-7220). The strains identified byaccession number of public culture collections are publicly availablefrom the depositories.

The organic acids which are present in the culture medium of theinvention are acetic acid, fumaric acid, maleic acid, malic acid,succinic acid, citric acid, a-ketoglutaric acid, lactic acid or gluconicacid. The organic acid may be present in the culture medium in an amountof l to g/dl, and may be present in the beginning of the fermentationperiod or it may be added gradually with or without a carbohydrate,while the fermentation proceeds. The pH of the culture medium rises asthe fermentation proceeds, and the pH can be adjusted by adding the freeorganic acid.

The following Table 1 shows that acetic acid when used as the carbonsource and for pH adjustment does not inactivate the protease produced.A culture medium containing the carbon source indicated in Table l, lg/dn soybean powder, 1.5 g/dl casein, 0.05 g/dl Kl-l PO and 0.2 g/dlCaCl -7H O, of pH 7.0 was inoculated with one liter seed culture ofBacillus subtilis AJ-3 168(FERM P-242) which had previously beenprepared in a medium having the same composition as that of test run No.l at 34 C. for 12 hours, and was cultured at 34 C. for 64 hours withaerating and with stirring. In the test run No. 3, acetic acid was addedto maintain the pH of the medium at 6.5 after 8 hours from theinoculation.

Protease activity was determined by a modification of Anthon-Hagiwarasmethod, in which a borate buffer solution at pH 9.5 was used at 37 C.for 10 minutes.

* pH was adjusted by adding acetic acid. **pH was adjusted with sulfuricacid. In test run Nos. 1 and 2, pH was not adjusted.

The culture medium should contain a source of assimilable nitrogen,inorganic salts and the usual organic nutrients. Suitable sources ofnitrogen include soybean cake, soybean powder, soybean extracts, milkcasein, milk whey, polypeptone, meat extracts and amino acid mixtures,as well as ammonium salts of mineral acids. The usual organic nutrientsare com steep liquor, peptone, yeast extracts and malt extracts.

In the known processes in which starch is used as the carbon source, astarch concentration of 7 to 10 g/d] can not be used,

because the medium is too viscous to be stirred for introducing air.According to the present invention, a large amount of organic acid canbe used and therefore a large amount of protease is produced.

The protease can be recovered from the culture broth by conventionalmethods. For example, bacterial cells are removed from the culture brothby filtration or by centrifuging, the protease isprecipitated by saltingout or by a non-solvent, and is isolated by filtration of centrifuging.

EXAMPLE 1 A culture medium of pH 7.0 containing 1.5 g/d] casein, 2.0g/dl Ajipro (trade name of water-soluble soy protein), 0.25 g/dl KH PO0.02 g/dl MgSO '7H O, 0.2 g/dl CaCl -2H O and 2 ml/ml Aji-Eki" (tradename of soybean protein hydrolyzate) was prepared, 30 liters of themedium was placed in a jarfermentor, and sterilized at 120 C. for 30minutes. The medium was adjusted to Ph 6.7 with NaOH solution,inoculated with a strain of bacteria as indicated in Table 2, andcultured at 34 C. for 64 hours with aerating and stirring.

During the cultivation, the pH of the medium was maintained at 6.7 byadding 50 percent acetic acid solution. Protease was produced in theculture broth as listed in Table TABLE 2 Protease Strain used Aceticacid produced used (g/dl) (unit/ml) Bacillus subtilis FERM P-242 7.25900 Bacillus sp NRRL 3-3699 9.8 7600 Bacillus sp NRRL 3-3700 l5.3 10300EXAMPLE 2 A culture medium containing 3 g/dl starch, l g/dl soybeanpowder, 1.5 g/dl casein, 0.05 g/dl KH PO and 0.2 g/dl CaCl -7 H O wasprepared, the pH ofthe medium was adjusted to 7.2 with NaOH, and themedium was sterilized. The pH of the medium was adjusted to 7.0, 20liters of the medium was inoculated with Bacillus sp AJ-3208 (NRRLB-3700), and cultured at 34 C. for 64 hours with aerating and stirring.The pH of the medium was held below 6.1 by adding 30 percent malic acidsolution during the cultivation. The amount of malic acid consumed was 4g per milliliters medium.

The culture broth was found to contain 4,500 units of protease permilliliters.

EXAMPLE 3 A culture medium having the same composition as that ofExample 2 was inoculated with Bacillus subtilis FERM P-242, and culturedfor 64 hours. During the cultivation, gaseous ammonia was introduced tohold the pH of the medium to a minimum of 6.5, and 50 percent aceticacid solution was added to hold the pH to not more than 7.0. Acetic acidwas used in an amount of 5 g/dl. The culture broth obtained was found tocontain 5,200 units/ml of protease.

EXAMPLE 4 Organic acid anions in the form of the potassium salts listedin Table 3 were added to culture media having the same composition as inExample 2, and each medium was inoculated with Bacillus subrilis AJ-3168(FERM P-242), and cultured at 34 C. for 64 hours with aerating and withstirring. The results obtained are listed in Table 3.

A culture medium of pH 7.0 containing 1.0 g/dl casein, 1.0 gldl "Ajiprd,0.25 g/dl KH,PO,, 0.02 g/dl MgSO,-7H O, 0.2 g/dl CaCl '2H-,O and 2 ml/dlAji-Eki", was prepared and, liters of the medium was inoculated withCandida lipolylica AJ- 45S5(ATCC 16617), and cultured at 25 C. for 64hours with aerating and stirring. During the cultivation, the pH of themedium was maintained at 7.0 by adding 50 percent acetic acid solution.Acetic acid was used in an amount of 4.3 g/dl. The culture broth wasfound to contain 350 units/ml of weakly alkaline protease at pH 8.5.

EXAMPLE 6 A culture medium having the same composition as that ofExample 5 was prepared, the pH of the medium was adjusted to 3.0, andRhodoromla gluu'nis AJ-S 193(NRRL Y-7220) was cultured on the medium inthe same way as in Example 5. The pH of the medium was maintained at 3.0by adding 50 percent acetic acid solution. Acetic acid was used in anamount of 4.8 gldl after 64 hours.

The acid protease activity was determined in a lactic acid buffersolution of pH 3.0, at 37 C. for 10 minutes, and the culture broth wasfound to contain 850 units/ml of acid protease.

What we claim is:

1. A method of producing protease which comprises culturing amicroorganim of genus Bacillus, Candida or Rhodotoru- 1a on an aqueousculture medium including an organic acid as the principal source ofassimilable carbon, a source of assimilable nitrogen, necessaryinorganic salts, and organic nutrients under aerobic conditions, andrecovering the protease produced.

2. A method as set forth in claim 1, wherein said organic acid is aceticacid, malic acid, fumaric acid, maleic acid, succinic acid, citric acid,a-ketoglutaric acid, lactic acid or gluconic acid.

3. A method as set forth in claim 1, wherein said organic acid is aceticacid.

4. A method as set forth in claim 2, said bacterium is Bacillussubtilis, Candida lipolytlca or Rhodotorula glutinis.

5. A method as set forth in claim 1, wherein said bacterium is Bacillussubtilis FERM P-242, Bacillus .rp NRRL B-3699, Bacillus sp NRRL 13-3700,Candida lipolytica ATCC 16617, Candida lipolytica ATCC 16618 orRhodotorula glutinis NRRL Y-7220.

2. A method as set forth in claim 1, wherein said organic acid is aceticacid, malic acid, fumaric acid, maleic acid, succinic acid, citric acid,Alpha -ketoglutaric acid, lactic acid or gluconic acid.
 3. A method asset forth in claim 1, wherein said organic acid is acetic acid.
 4. Amethod as set forth in claim 2, said bacterium is Bacillus subtilis,Candida lipolytica or Rhodotorula glutinis.
 5. A method as set forth inclaim 1, wherein said bacterium is Bacillus subtilis FERM P-242,Bacillus sp NRRL B-3699, Bacillus sp NRRL B-3700, Candida lipolyticaATCC 16617, Candida lipolytica ATCC 16618 or Rhodotorula glutinis NRRLY-7220.